1. Field of the Invention
The present invention relates to a new process for effecting peptide synthesis. More specifically, it relates to a new process for preparing an amino acid fluoride, and also to the use thereof in peptide synthesis. Furthermore, the present invention relates to new reagents for preparing the amino acid fluorides.
2. Background of the Invention
Amino acid fluorides have been shown to be a convenient and highly efficient reagent for both solution and solid phase peptide synthesis. When used in peptide synthesis, the peptide formed therefrom is produced in high yield and relatively pure form, with minimal racemization. Furthermore, it has been shown that there are many other advantages associated with the use of amino acid fluorides in peptide synthesis. For example, the acid fluorides allow the syntheses of peptides which incorporate highly-hindered amino acids, such as .alpha.-amino isobutyric acid (Aib) and .alpha.-ethylalanine (e.g., isovaline, Iva), and the like. Furthermore, amino acid fluorides exhibit several advantages relative to other amino acid halides, e.g., amino acid chlorides or bromides, in the coupling reaction and formation of peptides. For example, unlike the other amino acid halides, amino acid fluorides can accommodate t-butyl side chain protecting groups. Moreover, conversion to the corresponding oxazolone in the presence of t-organic base does not occur, thus avoiding the danger of racemization. Furthermore, the coupling reactions occur readily in the complete absence of an organic base, again avoiding possible racemization.
Moreover, another advantage of amino acid fluorides is that they are easily synthesized from the corresponding amino acid and are isolable in crystalline form. They are generally stable and have a long shelf life.
In view of these advantages, it is highly desirable to utilize amino acid fluorides in peptide synthesis. Thus, peptide synthesis can be effected by first preparing a N-.alpha.-amino protected amino acid fluoride and then utilizing this amino acid fluoride as a coupling agent to produce the desired peptide.
Unfortunately, when employing FMOC amino acid fluorides in practical peptide synthesis, difficulties were encountered in the case of two amino acids, arginine and histidine. In the latter case, while FMOC--His (Trt)--F has been synthesized and used in coupling reactions, its long term shelf stability is in doubt. For sulfonamide-protected arginine derivatives (e.g., FMOC--Arg (Pbf)--OH or FMOC--Arg--(Pmc)--OH)), the corresponding acid fluorides could not be synthesized due to their facile cyclization to the corresponding lactam.
Thus, investigations were conducted to improve the efficiency thereof and to overcome these problems. It was felt that the efficiency of the overall process would be enhanced if the amino acid fluoride were produced in situ. Thus, Carpino et al., as described in JACS 95, 117, 5401, developed a new uronium-style reagent TFFH, 1, which ##STR1##
has been shown to act as a coupling reagent which acts via in situ conversion to an acid fluoride: ##STR2##
Although a useful technique, especially since TFFH is relatively inexpensive, a disadvantage of this method is the need to use a basic reagent, such as N,N-diisopropylethylamine (DIEA) in the activation step (Eq. 1). Indeed, the speed of conversion of the acid to the acid fluoride increases with the number of equivalents of DIEA used (1 eq&lt;&lt;2 eqs&lt;3 eqs&lt;4 eqs).
Although this technique was more efficient than methods heretofore used in peptide coupling, scientific investigations were conducted to improve upon this reagent. It was believed that the in situ process for the preparation of protected amino acid fluorides and the overall process of peptide synthesis would be improved if a method could be found to generate amino acid fluorides in situ without the presence of a basic reagent. The present inventors have found such a method. Moreover, the present in situ process overcomes the difficulties discussed hereinabove with histidine and arginine.